Listeria monocytogenes (Lm) is a food-borne facultative intracellular bacterial pathogen that has been responsible for some of the deadliest contaminated food outbreaks in U.S. history. While disease in healthy individuals is usually limited to gastroenteritis, serious invasive disease occurs in susceptible populations that include immunocompromised individuals, pregnant women, neonates, and the elderly. Lm infections are commonly associated with meningitis, meningoencephalitis, brain abscesses, cardiac infections, septicemia, or stillbirth and abortion in pregnant women. Invasive disease resulting from Lm infection has a mortality rate that ranges from approximately 20% to up to 66% or higher despite antibiotic treatment, and surviving individuals often suffer from debilitating neurological sequelae. New effective strategies for successful treatment of Lm invasive infections are urgently needed to limit the severity of disease and the serious post-infection sequelae. In addition, a broader understanding of the molecular adaptations that enable Lm to transition from soil saprophyte to pathogen may facilitate better assessment of environmental reservoirs of disease. The focus of this proposal is on deciphering how a novel bacterial peptide pheromone-based signaling system enables a soil bacterium to transition into life within the mammalian cytosol. Central to the Lm soil-to-cytosol transition is the ability of the bacterium to sense its location within the vacuoles of infected host cells and express gene products that promote vacuole lysis and bacterial escape into the cytosol where replication occurs. We have identified a bacterial peptide pheromone that enhances Lm escape from host vacuoles and which is required for bacterial virulence in animals. The working hypothesis of this R21 proposal is that the pPplA peptide pheromone enables Lm to sense the spatial confines of the vacuole and induce the expression of gene products necessary for vacuole escape and cytosolic replication. This proposal will use a combination of genetic, biochemical, and in vivo approaches to functionally decipher the contributions of the pPplA peptide pheromone to bacterial virulence. Aim 1 will define the expression patterns and components of the pPplA signaling pathway, and Aim 2 will determine the mechanism by which pPplA enhances bacterial escape from host vacuoles. The ultimate goal of this proposal will be to elucidate the molecular pathways that promote Lm survival within host cells and the transformation of a soil dweller into a cell invader.